The ?E cell envelope-sensing pathway is a key regulatory system used by bacteria to maintain cell envelope integrity. ?E is essential for viabiliy of several important bacterial pathogens and required for virulence of others. ?E activity is regulated by two major signaling systems that integrate distinct signals, cell envelope stress and nutrient limitation. In response to envelope stress, ?E is activated by regulated proteolysis of it dedicated antisigma factor. During nutrient limitation ?E is activated by the alarmone ppGpp, in the absence of apparent envelope stress, preparing the envelope for survival in advance. The extent of activation by each system serves to set the overall activity of ?E in the cell, matching t to cellular needs. In addition to activating ?E, ppGpp activates other alternative sigma factors and stress pathways, while repressing genes required for rapid growth. In effect, ppGpp mediates a switch from a transcriptional program for rapid growth to one optimized for stress survival. The mechanism by which ppGpp regulates ?E activity is not wel understood. Such knowledge is critical for our understanding of how this important and highly conserved alternative sigma factor responds to changing environments to maintain cell envelope integrity. The objective of this proposal is to determine the mechanism(s) by which ?E activity is regulated by the global regulator, ppGpp, thereby coordinating envelope and cytoplasmic responses to starvation. The central hypothesis of the proposed research is that ppGpp, in conjunction with the coregulator DksA and potentialy other factors, controls ?E activity by directly activating ?E holoenzyme (E?E) and indirectly by increasing the amount of E?E through its influence on the competition among sigma factors for core RNA polymerase (RNAP). This hypothesis will be addressed through three specific aims. (1) Define the biochemical properties of transcription initiation by E?E. ?E is a member of a highly conserved, widely distributed class of alternative sigma factors that lack several conserved regions and amino acids known to be important for specific steps in transcription initiation by other sigma factors. How these differences alter transcription initiation has not been investigated. The fundamental parameters of transcription initiation by E?E must be established before the influence of pGp can be assessed. (2) Elucidate the mechanism by which ppGpp and DksA regulate E?E activity. Since activation of E?70 and alternative sigma factors by ppGpp/DksA is not well understood, results from the proposed work will have a strong positive impact on the overall understanding of the biochemical basis of transcriptional activation by these global regulators. (3) Determine how ppGpp regulates ?E activity independently of DksA. Preliminary work suggests that a novel mechanism of regulation of ?E by ppGpp exists that cannot be explained by current models, which require DksA to act with ppGpp. This novel regulatory strategy will be explored. Because both the ?E and ppGpp systems are critical for virulence in important bacterial pathogens, a better understanding of the intersection of these responses may lead to new avenues for drug discovery and vaccine design.